KR20180003322A - Composition for promoting angiogenic activity comprising exosome-mimetic nanovesicles derived from adult stem cells - Google Patents
Composition for promoting angiogenic activity comprising exosome-mimetic nanovesicles derived from adult stem cells Download PDFInfo
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- KR20180003322A KR20180003322A KR1020160082981A KR20160082981A KR20180003322A KR 20180003322 A KR20180003322 A KR 20180003322A KR 1020160082981 A KR1020160082981 A KR 1020160082981A KR 20160082981 A KR20160082981 A KR 20160082981A KR 20180003322 A KR20180003322 A KR 20180003322A
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Abstract
Description
본 명세서에는 성체줄기세포 유래의 엑소좀-모사 나노베지클을 유효성분으로 포함하는 혈관 신생 촉진용 조성물 및 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클을 제조하는 방법이 개시된다.In this specification, a composition for promoting angiogenesis, which comprises an exosome-simna nano bezle derived from adult stem cells as an effective ingredient, and a method for producing the exosome-simna nano beads from the adult stem cells are disclosed.
혈관 신생(angiogenesis)이란 새로운 혈관이 생성되는 과정을 말하며, 정상적인 생체 조건에서는 드물게 일어나지만, 배발생, 황체 형성 또는 상처 치료 과정에서는 반드시 수반되는 과정이다. Angiogenesis is the process by which new blood vessels are created, which rarely occurs under normal in vivo conditions, but is an integral part of the process of embryogenesis, luteinization or wound healing.
혈관 신생이 일어나는 과정은 일반적으로 혈관 신생 촉진 인자의 자극에 의하여 프로테아제로 인한 혈관 기저막의 분해, 혈관 내피세포의 이동, 증식 및 혈관 내피세포의 분화에 의한 관강의 형성으로 혈관이 재구성되어 새로운 모세혈관이 생성되는 것으로 이루어진다. 혈관 생성 과정은 생장인자, 사이토카인, 지질대사물질 및 지혈 단백질의 잠재성 단편 등 여러 종류의 촉진 인자와 억제 인자에 의해 엄격하게 통제되는 것으로 알려져 있다.In the process of angiogenesis, blood vessels are reconstituted by stimulation of angiogenesis promoting factors, resulting in degradation of blood vessel basement membrane due to protease, migration and proliferation of vascular endothelial cells, and formation of vessels by differentiation of vascular endothelial cells, Is generated. The process of angiogenesis is known to be tightly controlled by several types of promoters and inhibitors, including growth factors, cytokines, lipid metabolites and potential fragments of hemostatic proteins.
발생 및 상처 치유, 기관 형성에 중요한 과정인 혈관 생성이 제대로 일어나지 않을 경우 심각한 질환이 발생하게 된다. 예를 들어, 발생 단계에서 혈관 형성이 되지 않을 경우 태반의 미발달을 초래하여 유산의 원인이 될 수 있으며, 조직의 궤양 및 허혈의 발생이 기관의 기능 이상을 유발시키고 더 나아가 사망에 이르게 한다. 최근 식생활의 변화, 영양 상태의 개선과 노년 인구의 증가에 따라 동맥경화, 심근경색 및 협심증, 뇌경색, 급성사지허혈과 같은 다양한 심혈관계 질환이 성인 사망률의 수위를 차지하는 심각한 질환으로 대두되고 있으나 뚜렷한 치료법이 정립되지 않은 상태이며, 이러한 허혈성 질환을 치료하기 위해 신생 혈관 형성을 유도 또는 촉진하는 새로운 치료법 및 인자의 개발이 주목을 받고 있다(김덕경 et al, 대한내분비학회, 16(3), 328-338, 2001).If blood vessel formation, which is an important process in the development and wound healing and organ formation, does not occur properly, serious diseases will occur. For example, if blood vessels are not formed in the developmental stage, placenta may be undeveloped and cause abortion, and the occurrence of tissue ulcers and ischemia may cause dysfunction of the organs, leading to further death. Recent changes in dietary habits, nutritional status, and aging population have led to a variety of cardiovascular diseases, including arteriosclerosis, myocardial infarction and angina, cerebral infarction, and acute limb ischemia, And the development of new therapies and factors that induce or promote neovascularization to treat such ischemic diseases has been attracting attention (Kim, Duk-Kyung et al., Korean Endocrinology Society, 16 (3), 328-338 , 2001).
혈관 신생을 이용한 생체 질환의 치료를 혈관 신생 치료라 하며, 혈관내피 성장인자(VEGF)와 같은 혈관 신생 촉진 인자가 중증의 국소 빈혈을 위한 치료제로 사용되고 있으나, 상기 인자들의 분리 및 정제가 어려우며, 고가이므로 임상 적용에 어려움이 있고, 혈관성 조직 복구에 의해 증상이 개선될 수 있는 질환의 치료를 위해 보다 효과적이고 새로운 인자들의 발굴이 지속적으로 요구되고 있는 실정이다.Treatment of vascular diseases using angiogenesis is called angiogenesis therapy, and angiogenesis promoting factors such as vascular endothelial growth factor (VEGF) are used as therapeutic agents for severe ischemia, but it is difficult to separate and purify the above factors, Therefore, there is a continuing need to find more effective and novel agents for the treatment of diseases in which clinical application is difficult and symptoms can be improved by vascular tissue repair.
한편, 줄기세포(stem cell)란 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 줄기세포는 분화능에 따라 만능 줄기세포(totipotent stem cell), 전분화능 줄기세포(pluripotent stem cells), 다분화능(다능성) 줄기세포(multipotent stem cells)로 분류할 수 있으며, 세포학적 유래에 따라 배아줄기세포와 성체줄기세포로 분류할 수 있다. 배아줄기세포는 착상 전 수정란이나 발생중인 태아 생식기 조직 등에서 유래하는 반면, 성체줄기세포는 성체 내에서 존재하는 각 기관, 예를 들면 골수, 뇌, 간, 췌장 등에서 유래한다.On the other hand, stem cells are undifferentiated cells that have the ability to self-replicate and have the ability to differentiate into two or more different types of cells. Stem cells can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells depending on their differentiation ability. According to their cytological origins, embryonic stem cells can be classified into embryonic stem cells, Stem cells and adult stem cells. Embryonic stem cells originate from pre-implantation embryos or from the developing fetal genital tissue, whereas adult stem cells originate from each organ present in the adult, such as bone marrow, brain, liver, pancreas, and the like.
중간엽 줄기세포가 혈관세포에 미치는 영향(신생 혈관 생성 및 억제 등)은 잘 알려져 있다(Front Neurosci., 24(7):194, 2013). 중간엽 줄기세포 유래의 엑소좀 또한 신생 혈관 생성 효과 및 심혈관 질환 예방 효과가 있음이 알려져 있으며, 이로 인해 중간엽 줄기세포 유래의 엑소좀은 재생의학 분야에서 새로운 치료 물질로서 각광을 받고 있다(Front Immunol., 4(5):370, 2014). 그러나, 중간엽 줄기세포 유래의 엑소좀은 분리 및 정제하기가 어렵고 수득률이 낮은 문제로 인해 그 활용이 어렵다.The effects of mesenchymal stem cells on vascular cells (neovascularization and inhibition) are well known (Front Neurosci., 24 (7): 194, 2013). It is known that exosome derived from mesenchymal stem cells also has neovascularization effect and cardiovascular disease prevention effect, and thus exosome derived from mesenchymal stem cells is in the spotlight as a new therapeutic substance in the field of regenerative medicine (Front Immunol , 4 (5): 370, 2014). However, exosomes derived from mesenchymal stem cells are difficult to separate and purify, and are difficult to utilize because of the low yield.
일 측면에서, 본 명세서는 성체줄기세포 유래의 엑소좀-모사 나노베지클을 유효성분으로 포함하는 혈관 신생 촉진용 조성물을 제공하는 것을 목적으로 한다.In one aspect, the present invention aims to provide a composition for promoting angiogenesis, which comprises, as an active ingredient, exosome-mimosa nano-beads derived from adult stem cells.
다른 측면에서, 본 명세서는 종래 성체줄기세포 유래의 엑소좀의 문제점을 개선시킨, 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클을 높은 수득률로 제조하는 방법을 제공하는 것을 목적으로 한다.In another aspect, the object of the present invention is to provide a method for producing the exosome-mimic nano-beadlet derived from adult stem cells at a high yield, which improves the problem of exosomes derived from adult stem cells in the past.
일 측면에서, 본 명세서에 개시된 기술은 성체줄기세포 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles)을 유효성분으로 포함하는 혈관 신생 촉진용 조성물을 제공한다.In one aspect, the technology disclosed herein provides a composition for promoting angiogenesis, which comprises exosome-mimetic nanovesicles derived from adult stem cells as an active ingredient.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 성체줄기세포를 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시킨 성체줄기세포의 여과물에 포함된 나노 크기의 베지클인 것일 수 있다.In one exemplary embodiment, the exosome-mimetic nano-beadlet is included in a filtrate of adult stem cells that sequentially pass adult stem cells through two or more different size membrane filters in the order of a larger filter to a smaller filter Sized nano-sized beads.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 30 내지 200 nm의 직경을 갖는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nanobubble may have a diameter of 30 to 200 nm.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 성체줄기세포의 여과물로부터 10% 및 50% 농도의 이오딕사놀을 이용한 밀도 구배 초원심분리 시, 10% 농도의 이오딕사놀과 50% 농도의 이오딕사놀 사이에 위치하는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nano-beadlets are obtained from a filtrate of adult stem cells in a density gradient ultracentrifugation using 10% and 50% concentrations of iodixanol at a concentration of 10% iodixanol Lt; RTI ID = 0.0 > 50% < / RTI > concentration.
예시적인 일 구현예에서, 상기 성체줄기세포는 골수, 제대혈, 혈액, 피부, 지방 및 태반으로 이루어진 군에서 선택되는 1 이상으로부터 유래된 중간엽 줄기세포일 수 있다.In one exemplary embodiment, the adult stem cells may be mesenchymal stem cells derived from one or more selected from the group consisting of bone marrow, umbilical cord blood, blood, skin, fat and placenta.
예시적인 일 구현예에서, 상기 성체줄기세포는 TNF-α를 처리하여 배양한 것일 수 있다.In an exemplary embodiment, the adult stem cells may be cultured by treating TNF-a.
예시적인 일 구현예에서, 상기 성체줄기세포는 passage 6까지 계대배양한 후 TNF-α를 처리하여 배양한 것일 수 있다.In an exemplary embodiment, the adult stem cells may be cultured by passaging to passage 6, followed by treatment with TNF-a.
예시적인 일 구현예에서, 상기 멤브레인 필터는 1 내지 10 ㎛ 크기의 평균 공극을 갖는 것일 수 있다.In an exemplary embodiment, the membrane filter may have an average pore size of 1 to 10 [mu] m.
예시적인 일 구현예에서, 상기 초원심분리는 100,000 ×g 이상에서 수행하는 것일 수 있다.In an exemplary embodiment, the ultracentrifugation may be performed at 100,000 x g or more.
예시적인 일 구현예에서, 상기 조성물은 혈관 내피세포의 이동을 유도 또는 증가시키는 것일 수 있다.In an exemplary embodiment, the composition may be one that induces or increases the migration of vascular endothelial cells.
예시적인 일 구현예에서, 상기 조성물은 혈관 신생이 요구되는 질환에서 혈관 신생을 촉진하는 것일 수 있다.In an exemplary embodiment, the composition may be to promote angiogenesis in a disease in which angiogenesis is desired.
예시적인 일 구현예에서, 상기 혈관 신생이 요구되는 질환은 상처, 화상, 궤양, 허혈, 동맥경화증, 협심증, 심근경색, 뇌혈관성 질환 및 탈모증으로 이루어진 군에서 선택되는 1 이상일 수 있다.In an exemplary embodiment, the disease for which angiogenesis is required may be one or more selected from the group consisting of wound, burn, ulcer, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease and alopecia.
예시적인 일 구현예에서, 상기 조성물은 약학 조성물 또는 식품 조성물일 수 있다.In an exemplary embodiment, the composition may be a pharmaceutical composition or a food composition.
다른 측면에서, 본 명세서에 개시된 기술은 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles)을 제조하는 방법으로서, (1) 성체줄기세포를 배양하는 단계; (2) 배양된 성체줄기세포를 수확하여 완충용액에 현탁시키는 단계; (3) 상기 현탁액을 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시켜 여과물을 제조하는 단계; 및 (4) 상기 여과물로부터 초원심분리를 통해 엑소좀-모사 나노베지클을 수득하는 단계;를 포함하는 성체줄기세포 유래의 엑소좀-모사 나노베지클을 제조하는 방법을 제공한다.In another aspect, the technique disclosed in the present invention is a method for producing exosome-mimetic nanobeicles derived from adult stem cells, comprising the steps of: (1) culturing adult stem cells; (2) harvesting the cultured adult stem cells and suspending them in a buffer solution; (3) passing the suspension through two or more size different membrane filters in order from a large filter to a small filter in order to produce a filtrate; And (4) obtaining an exosome-simulated nano bead by ultracentrifugation from the filtrate. The present invention also provides a method for producing an exosome-simulated nano beads from an adult stem cell.
일 측면에서, 본 명세서에 개시된 기술은 성체줄기세포 유래의 엑소좀-모사 나노베지클을 유효성분으로 포함하는 혈관 신생 촉진용 조성물을 제공하는 효과가 있다.In one aspect, the technique disclosed in this specification has an effect of providing a composition for promoting angiogenesis, which comprises, as an active ingredient, exosome-mimetic nanobasech derived from adult stem cells.
다른 측면에서, 본 명세서에 개시된 기술은 종래 성체줄기세포 유래의 엑소좀의 문제점을 개선시킨, 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클을 높은 수득률로 제조하는 방법을 제공하는 효과가 있다.In another aspect, the technique disclosed in this specification has an effect of providing a method of producing the exosome-mimic nano-beadlet derived from adult stem cells at a high yield, which improves the problem of exosomes derived from adult stem cells .
도 1은 본 명세서의 일 시험예에 따라 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 제조하는 공정을 나타낸 것이다.
도 2는 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 분석 결과를 나타낸 것이다. 도 2a는 엑소좀-모사 나노베지클의 형태, 도 2b는 엑소좀-모사 나노베지클의 크기를 나타낸다.
도 3은 종래 중간엽 줄기세포 유래의 엑소좀과 본 명세서에 따른 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 단밸질의 양 및 입자수를 비교한 결과를 나타낸 것이다.
도 4는 본 명세서의 일 시험예에 따른 Matrigel plug assay 흐름도를 나타낸 것이다.
도 5는 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 효과를 나타낸 것이다.
도 6은 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 면역세포 침윤 효과를 나타낸 것이다.
도 7은 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 내피세포 증식 및 이동에 미치는 영향을 나타낸 것이다.
도 8은 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 대식세포 이동에 미치는 영향을 나타낸 것이다.
도 9는 본 명세서의 일 시험예에 따라 제조된 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클로 처리된 대식세포가 내피세포 이동에 미치는 영향을 나타낸 것이다.FIG. 1 shows a process for preparing exosome-mimetic nanobeads from mesenchymal stem cells according to a test example of the present invention.
Fig. 2 shows the results of analysis of exosome-simulated nano-beads from mesenchymal stem cells prepared according to one test example of the present specification. FIG. 2A shows the shape of exosome-mimosa nano-bezacle, and FIG. 2B shows the size of exosome-mimosa nano-bezacle.
FIG. 3 shows the results of comparing the amount and number of single-stranded proteins of exo-soma-derived naturally occurring exosome-derived mesenchymal stem cells derived from mesenchymal stem cells according to the present invention.
4 is a flow chart of a Matrigel plug assay according to one test example of the present specification.
5 shows the neovascularization effect of exosome-mimosa nano-beads derived from mesenchymal stem cells prepared according to one test example of this specification.
Fig. 6 shows the effect of the exosome-simulated nano-beadlets derived from mesenchymal stem cells prepared according to one test example of the present invention on immune cell infiltration.
FIG. 7 shows the effects of mesenchymal stem cells derived from a mesenchymal stem cell-derived exosome-simulated nano-beads on endothelial cell proliferation and migration according to a test example of this specification.
Fig. 8 shows the effect of mesenchymal stem cell-derived exosome-simulated nano-beads prepared according to one test example of the present invention on macrophage migration.
Fig. 9 shows the effect of mesenchymal stem cell-derived exosome-simulated nanobegycle treated macrophages prepared according to one test example of the present invention on endothelial cell migration.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 명세서는 종래 성체줄기세포 유래의 엑소좀이 갖는 문제점을 개선하여 성체줄기세포 유래의 엑소좀-모사 나노베지클을 높은 수득률로 제조하였고, 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클이 우수한 신생 혈관 촉진 효과가 있음을 확인하였다.The present invention improves the problems of exosomes derived from adult stem cells in the past to produce exosome-mimosa nano beads derived from adult stem cells at a high yield, and the exosome-mimosa nano beads derived from adult stem cells And it was confirmed that there was an excellent effect of stimulating new blood vessels.
일 측면에서, 본 명세서에 개시된 기술은 성체줄기세포 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles)을 유효성분으로 포함하는 혈관 신생 촉진용 조성물을 제공한다.In one aspect, the technology disclosed herein provides a composition for promoting angiogenesis, which comprises exosome-mimetic nanovesicles derived from adult stem cells as an active ingredient.
본 명세서에서 "유효성분"은 단독으로 목적으로 하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체 등과 함께 목적으로 하는 활성을 나타낼 수 있는 성분을 의미한다.As used herein, the term "active ingredient" refers to a component that can exhibit the desired activity alone, such as a carrier that exhibits the desired activity alone or is itself inactive.
본 명세서에서 "엑소좀-모사 나노베지클"은 성체줄기세포로부터 얻은 나노 크기의 베지클을 의미하는 것으로서, 세포외 소낭인 엑소좀과 유사한 나노 크기를 갖는 베지클을 의미한다.As used herein, the term "exosome-sima nano-vesicle" refers to a nano-sized vesicle obtained from adult stem cells, and refers to a vesicle having a nano-size similar to the extracellular vesicle exosome.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 성체줄기세포를 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시킨 성체줄기세포의 여과물에 포함된 나노 크기의 베지클인 것일 수 있다.In one exemplary embodiment, the exosome-mimetic nano-beadlet is included in a filtrate of adult stem cells that sequentially pass adult stem cells through two or more different size membrane filters in the order of a larger filter to a smaller filter Sized nano-sized beads.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 30 내지 200 nm의 직경을 갖는 것일 수 있다. 더욱 구체적으로, 상기 엑소좀-모사 나노베지클은 30 nm 이상, 32 nm 이상, 34 nm 이상, 36 nm 이상, 38 nm 이상, 40 nm 이상, 42 nm 이상, 44 nm 이상, 46 nm 이상, 48 nm 이상 또는 50 nm 이상이면서, 200 nm 이하, 190 nm 이하, 180 nm 이하, 170 nm 이하, 160 nm 이하, 150 nm 이하, 140 nm 이하, 130 nm 이하, 120 nm 이하, 110 nm 이하, 100 nm 이하, 95 nm 이하, 90 nm 이하, 85 nm 이하, 80 nm 이하, 75 nm 이하 또는 70 nm 이하의 직경을 갖는 것일 수 있다. 예컨대, 상기 엑소좀-모사 나노베지클은 30 내지 100 nm, 40 내지 80 nm, 50 내지 100 nm, 또는 50 내지 80 nm의 직경을 갖는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nanobubble may have a diameter of 30 to 200 nm. More specifically, the exosome-mimetic nanobegicle may be at least 30 nm, at least 32 nm, at least 34 nm, at least 36 nm, at least 38 nm, at least 40 nm, at least 42 nm, at least 44 nm, at least 46 nm, at least 48 nm or more, or 50 nm or more, and 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, Or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, or 70 nm or less. For example, the exosome-simulated nanobase may have a diameter of 30 to 100 nm, 40 to 80 nm, 50 to 100 nm, or 50 to 80 nm.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 40 내지 55 nm의 평균 직경을 갖는 것일 수 있다. 더욱 구체적으로, 상기 엑소좀-모사 나노베지클은 40 nm 이상, 42 nm 이상, 44 nm 이상, 46 nm 이상, 48 nm 이상 또는 50 nm 이상이면서, 55 nm 이하, 54 nm 이하, 53 nm 이하, 52 nm 이하, 51 nm 이하 또는 50 nm 이하의 평균 직경을 갖는 것일 수 있다. 예컨대, 상기 엑소좀-모사 나노베지클은 42 내지 53 nm, 46 내지 52 nm, 48 내지 52 nm, 또는 50 nm의 평균 직경을 갖는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nano-beads may have an average diameter of 40 to 55 nm. More specifically, the exosome-mimetic nanobeads may have a specific surface area of 40 nm or more, 42 nm or more, 44 nm or more, 46 nm or more, 48 nm or more, 50 nm or more, 55 nm or less, 54 nm or less, 52 nm or less, 51 nm or less, or 50 nm or less. For example, the exosome-simulated nano beads may have an average diameter of 42-53 nm, 46-52 nm, 48-52 nm, or 50 nm.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 성체줄기세포의 여과물로부터 10% 및 50% 농도의 이오딕사놀을 이용한 밀도 구배 초원심분리 시, 10% 농도의 이오딕사놀과 50% 농도의 이오딕사놀 사이에 위치하는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nano-beadlets are obtained from a filtrate of adult stem cells in a density gradient ultracentrifugation using 10% and 50% concentrations of iodixanol at a concentration of 10% iodixanol Lt; RTI ID = 0.0 > 50% < / RTI > concentration.
예시적인 일 구현예에서, 상기 성체줄기세포는 자가 또는 동종 유래의 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함하는 임의 유형의 동물로부터 유래할 수 있다.In an illustrative embodiment, the adult stem cells may be autologous or allogeneic stem cells and may be derived from any type of animal, including human and non-human mammals.
예시적인 일 구현예에서, 상기 성체줄기세포는 골수, 제대혈, 혈액, 피부, 지방 및 태반으로 이루어진 군에서 선택되는 1 이상으로부터 유래된 중간엽 줄기세포일 수 있다.In one exemplary embodiment, the adult stem cells may be mesenchymal stem cells derived from one or more selected from the group consisting of bone marrow, umbilical cord blood, blood, skin, fat and placenta.
예시적인 일 구현예에서, 상기 성체줄기세포는 TNF-α를 처리하여 배양한 것일 수 있다.In an exemplary embodiment, the adult stem cells may be cultured by treating TNF-a.
예시적인 일 구현예에서, 상기 성체줄기세포는 passage 6까지 계대배양한 후 TNF-α를 처리하여 배양한 것일 수 있다.In an exemplary embodiment, the adult stem cells may be cultured by passaging to passage 6, followed by treatment with TNF-a.
예시적인 일 구현예에서, 상기 멤브레인 필터는 1 내지 10 ㎛ 크기의 평균 공극을 갖는 것일 수 있다.In an exemplary embodiment, the membrane filter may have an average pore size of 1 to 10 [mu] m.
예시적인 일 구현예에서, 상기 멤브레인 필터는 크기가 다른 세 종류의 필터로 구성될 수 있으며, 예컨대 1 내지 2 ㎛ 크기의 평균 공극을 갖는 필터, 4 내지 6 ㎛ 크기의 평균 공극을 갖는 필터, 및 9 내지 10 ㎛ 크기의 평균 공극을 갖는 필터로 구성될 수 있다. In an exemplary embodiment, the membrane filter may be composed of three different sized filters, such as a filter having an average pore size of 1 to 2 mu m, a filter having an average pore size of 4 to 6 mu m, And a filter having an average pore size of 9 to 10 mu m in size.
예시적인 일 구현예에서, 상기 초원심분리는 100,000 ×g 이상, 구체적으로 100,000 내지 200,000 ×g, 또는 100,000 내지 150,000 ×g, 또는 150,000 내지 200,000 ×g에서 수행하는 것일 수 있다.In an exemplary embodiment, the ultracentrifugation may be performed at 100,000 x g or more, specifically 100,000 to 200,000 x g, or 100,000 to 150,000 x g, or 150,000 to 200,000 x g.
예시적인 일 구현예에서, 상기 엑소좀-모사 나노베지클은 예컨대, 표적 세포에서 원하는 기능을 효율적으로 수행할 수 있도록 막 성분이 화학적 또는 물리적으로 변형된 것일 수 있다. 예를 들어, 엑소좀-모사 나노베지클의 막 성분이 티올기(-SH) 또는 아민기(-NH2)를 이용하여 화학적인 방법으로 변형되거나, 엑소좀-모사 나노베지클에 표적유도 물질, 세포막융합 물질, 폴리에틸렌글리콜을 화학적으로 결합시켜 막 이외의 성분을 더 포함하는 것일 수 있다.In an exemplary embodiment, the exosome-simulated nano-bezacl may be one in which the membrane component is chemically or physically modified so as to efficiently perform the desired function in the target cell. For example, if the membrane component of the exosome-sima nano beads is modified by a chemical method using a thiol group (-SH) or an amine group (-NH 2 ), or the exosome- , A cell membrane fusion material, and polyethylene glycol, so as to further contain components other than the membrane.
예시적인 일 구현예에서, 상기 조성물은 혈관내피세포의 이동을 유도 또는 증가시키는 것일 수 있다.In an exemplary embodiment, the composition may be one that induces or increases the migration of vascular endothelial cells.
예시적인 일 구현예에서, 상기 조성물은 혈관 신생이 요구되는 질환에서 혈관 신생을 촉진하는 것일 수 있다.In an exemplary embodiment, the composition may be to promote angiogenesis in a disease in which angiogenesis is desired.
예시적인 일 구현예에서, 상기 혈관 신생이 요구되는 질환은 상처, 화상, 궤양, 허혈, 동맥경화증, 협심증, 심근경색, 뇌혈관성 질환 및 탈모증으로 이루어진 군에서 선택되는 1 이상일 수 있다.In an exemplary embodiment, the disease for which angiogenesis is required may be one or more selected from the group consisting of wound, burn, ulcer, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease and alopecia.
또 다른 측면에서, 본 명세서에 개시된 기술은 혈관 신생에 유효한 양의 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클을 유효성분으로 포함하는 조성물을 이를 필요로 하는 대상에게 투여하는 것을 포함하는, 혈관 신생 촉진 방법을 제공한다.In another aspect, the art disclosed herein relates to a method of treating a subject suffering from angiogenesis, comprising administering to a subject in need thereof a composition comprising, as an active ingredient, an exosome-simulated nanobasech from an adult stem cell- Thereby providing a method for promoting angiogenesis.
예시적인 일 구현예에서, 상기 조성물은 동결건조된 제형일 수 있다. 상기 조성물은 즉시 사용 가능하도록(ready-to-use) 밀봉된 포장재 또는 포장 용기에 담긴 동결건조된 제형일 수 있다. In an exemplary embodiment, the composition may be a lyophilized formulation. The composition may be a ready-to-use sealed package or a lyophilized formulation in a packaging container.
본 명세서는 또한 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클을 유효성분으로 포함하고 동결건조된 제형을 갖는 조성물; 및 멸균수 또는 정제수;를 포함하는 혈관 신생 촉진용 키트를 제공한다. 상기 키트는 즉시 사용 가능하도록(ready-to-use) 밀봉된 포장재 또는 포장 용기에 담긴 것일 수 있다.The present invention also relates to a composition comprising the above-described adult stem cell-derived exosome-simulated nanobasech as an active ingredient and having a lyophilized formulation; And a kit for promoting angiogenesis, which comprises sterile water or purified water. The kit may be in a ready-to-use sealed packaging or packaging container.
예시적인 일 구현예에 따르면, 상기 조성물은 약학 조성물인 것일 수 있다.According to one exemplary embodiment, the composition may be a pharmaceutical composition.
상기 약학 조성물은 엑소좀-모사 나노베지클 이외에 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 약제학적 보조제 및 기타 치료적으로 유용한 물질을 추가로 함유할 수 있으며, 통상적인 방법에 따라 다양한 경구 투여제 또는 비경구 투여제 형태로 제형화할 수 있다.The pharmaceutical composition may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances in addition to the exosome-simna nano bezel , And may be formulated into various oral or parenteral dosage forms according to conventional methods.
상기 경구 투여제는 예를 들면, 정제, 환제, 경질 및 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 분제, 산제, 세립제, 과립제, 펠렛제 등이 있으며, 이들 제형은 유효성분 이외에 계면 활성제, 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로오스 및 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및 폴리에틸렌 글리콜)를 함유할 수 있다. 정제는 또한 마그네슘 알루미늄 실리케이트, 전분페이스트, 젤라틴, 트라가칸스, 메틸셀룰로오스, 나트륨 카복시메틸셀룰로오스 및 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제, 흡수제, 착색제, 향미제, 및 감미제 등의 약제학적 첨가제를 함유할 수 있다. 상기 정제는 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다.Examples of the oral administration agent include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, powders, granules, granules and pellets, , Diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols) . The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be prepared by conventional mixing, granulating or coating methods.
또한, 상기 비경구 투여 형태로는 경피 투여형 제형일 수 있으며, 예를 들어 주사제, 점적제, 연고, 로션, 겔, 크림, 스프레이, 현탁제, 유제, 좌제(坐劑), 패취 등의 제형일 수 있으나, 이에 제한되는 것은 아니다.In addition, the parenteral administration forms may be transdermal dosage forms, and may be formulations such as injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, But is not limited thereto.
상기 유효성분의 투여량 결정은 통상의 기술자의 수준 내에 있으며, 약물의 1일 투여 용량은 투여하고자 하는 대상의 진행 정도, 발병 시기, 연령, 건강상태, 합병증 등의 다양한 요인에 따라 달라지지만, 성인을 기준으로 할 때 일 측면에서 상기 조성물 1 ㎍/kg 내지 200 mg/kg, 다른 일 측면에서 50 ㎍/kg 내지 50 mg/kg을 1일 1 내지 3회 분할하여 투여할 수 있으며, 상기 투여량은 어떠한 방법으로도 본 발명의 범위를 한정하는 것이 아니다.The dosage of the active ingredient is within the level of ordinary skill in the art and the daily dose of the drug depends on various factors such as the degree of progress of the subject to be administered, the age of onset, age, health condition, The composition may be administered in an amount of 1 / / kg to 200 mg / kg in one aspect, and 50 / / kg to 50 mg / kg in another aspect, divided into 1 to 3 times a day, Are not intended to limit the scope of the invention in any way.
예시적인 일 구현예에 따르면, 상기 조성물은 식품 조성물인 것일 수 있다.According to one exemplary embodiment, the composition may be a food composition.
상기 식품 조성물은 액상 또는 고체 상태의 제형일 수 있고, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용될 수 있다. 각 제형의 식품 조성물은 유효성분 이외에 해당 분야에서 통상적으로 사용되는 성분들을 제형 또는 사용 목적에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있으며, 다른 원료와 동시에 적용할 경우 상승 효과가 일어날 수 있다.The food composition may be in a liquid or solid form and may be in the form of, for example, various foods, beverages, gums, tea, vitamin complexes, health supplements and the like and may be in the form of powders, granules, tablets, capsules or beverages . The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients.
본 명세서에 개시된 유효성분 외에 함유할 수 있는 액체 성분에는 특별한 제한점이 없으며, 통상의 음료와 같이 여러가지 향미제 또는 천연 탄수화물 등을 추가성분으로 포함할 수 있다. 상기 천연 탄수화물의 예로는 모노사카라이드, 포도당, 과당 등의 디사카라이드, 말토스, 슈크로스 등의 폴리사카라이드, 덱스트린, 시클로덱스트린 등의 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당 알코올 등이 있다. 상기의 향미제로는 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(예를 들어 사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 명세서에 개시된 조성물 100 ml 당 일반적으로 약 1 내지 20 g, 일 측면에서 약 5 내지 12 g일 수 있다.There are no particular limitations on the liquid components that can be contained in addition to the active ingredients disclosed in this specification, and may include various flavoring agents or natural carbohydrates such as ordinary beverages as additional ingredients. Examples of the natural carbohydrate include common saccharides such as disaccharides such as monosaccharide, glucose and fructose, polysaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol . The above flavoring agents can be advantageously used as natural flavorings (tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (for example, saccharin, aspartame etc.) The ratio of the natural carbohydrate may be generally about 1 to 20 g, and in one aspect about 5 to 12 g per 100 ml of the composition disclosed herein.
상기 식품 조성물은 일 측면에서 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그 염, 알긴산 및 그 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있다. 다른 측면에서 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 상기 성분들은 독립적으로 또는 조합하여 사용될 수 있다. 상기 첨가제의 비율은 다양할 수 있으나, 본 명세서에 개시된 조성물 100 중량부 당 0.001 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In one aspect, the food composition may include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. On the other hand, flesh for the production of natural fruit juices and vegetable beverages. These components can be used independently or in combination. The proportion of the additive may vary, but is generally selected in the range of from 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.
또 다른 측면에서, 본 명세서는 상기 성체줄기세포 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles)을 제조하는 방법으로서, (1) 성체줄기세포를 배양하는 단계; (2) 배양된 성체줄기세포를 수확하여 완충용액에 현탁시키는 단계; (3) 상기 현탁액을 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시켜 여과물을 제조하는 단계; 및 (4) 상기 여과물로부터 초원심분리를 통해 엑소좀-모사 나노베지클을 수득하는 단계;를 포함하는 성체줄기세포 유래의 엑소좀-모사 나노베지클을 제조하는 방법을 제공한다.In another aspect, the present invention provides a method for producing exosome-mimetic nanovesicles derived from adult stem cells, comprising the steps of: (1) culturing adult stem cells; (2) harvesting the cultured adult stem cells and suspending them in a buffer solution; (3) passing the suspension through two or more size different membrane filters in order from a large filter to a small filter in order to produce a filtrate; And (4) obtaining an exosome-simulated nano bead by ultracentrifugation from the filtrate. The present invention also provides a method for producing an exosome-simulated nano beads from an adult stem cell.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
시험예 1. 인간 중간엽 줄기세포(Human mesenchymal stem cells, hMSCs) 유래의 Experimental Example 1. Experimental Example 1: Human mesenchymal stem cells (hMSCs) derived from human mesenchymal stem cells 엑소좀Exosome -모사 나노베지클(- Mosaic nano bezle ( exosomeexosome -mimetic -mimetic nanovesiclesnanovesicles , , NVsNVs )의 제조 )
본 시험예에서는 성체줄기세포의 일종인 골수 유래 중간엽 줄기세포를 이용하여 엑소좀-모사 나노베지클을 제조하였다. 상기 중간엽 줄기세포는 전체 골수 세포의 0.01%를 차지하고 있으며, 연골세포, 골격세포, 신경세포 등으로 분화할 수 있는 다능성(multipotency) 세포이다.In this test example, bone marrow-derived mesenchymal stem cells, a type of adult stem cells, were used to prepare exosome-simulated nano-beads. The mesenchymal stem cells account for 0.01% of the total bone marrow cells and are pluripotent cells capable of differentiating into chondrocytes, skeletal cells, neurons, and the like.
인간 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 제조하기 위해서, 세포를 passage 6까지 배양하고 신생 혈관 생성 효과를 증가시키기 위해서, TNF-α(20 ng/mL)를 처리하여 12시간 동안 추가로 배양하였다. 이후, PBS buffer로 세포를 두 번 washing한 후, 멸균된 cell scrapper를 이용하여 세포를 떼어내고 600 ×g, 5분 조건에서 세포를 침전시켰다. 침전된 세포는 PBS buffer로 재현탁시킨 후, 10 μm, 5 μm, 1 μm의 평균 공극 크기를 갖는 멤브레인 필터를 각각 5번씩 순차적으로 통과시켜 나노 크기의 엑소좀-모사 나노베지클을 제조하였다. 또한, 순수한 엑소좀-모사 나노베지클만을 분리하기 위해서, 튜브에 이오딕사놀(iodixanol) 50%, 10% 및 샘플을 아래에서부터 쌓고 밀도 구배 초원심분리(density gradient ultracentrifugation)를 100,000 ×g, 2시간, 4 ℃ 조건에서 수행하였다. 10%와 50% 이오딕사놀 사이에 존재하는 순수한 엑소좀-모사 나노베지클 층을 모으고, 버퍼 교환을 위해 HBS buffer로 희석하여 초원심분리를 100,000 ×g, 2시간, 4 ℃ 조건에서 수행하였다(도 1 참조).To prepare exosome-simulated nano-beads from human mesenchymal stem cells, TNF-α (20 ng / mL) was treated for 12 hours to increase the neovascularization effect And further cultured. After washing the cells twice with PBS buffer, the cells were detached using a sterilized cell scraper and the cells were settled at 600 × g for 5 minutes. The precipitated cells were resuspended in PBS buffer, and then membrane filters with average pore sizes of 10 μm, 5 μm, and 1 μm were sequentially passed through the membrane filters five times each to produce nano-sized exosome-mimic nano-beads. In order to isolate pure exosome-mimanobanzyme only, 50% of iodixanol, 10% of sample, and density gradient ultracentrifugation were added at 100,000 × g, 2 Hour, 4 < 0 > C. The pure exosomal-mimosa nano-bequick layer present between 10% and 50% iodixanol was pooled, diluted with HBS buffer for buffer exchange, and ultracentrifuged at 100,000 x g for 2 hours at 4 ° C (See Fig. 1).
시험예 2. 인간 중간엽 줄기세포(Human mesenchymal stem cells, hMSCs) 유래의 Test Example 2. Experimental Example 1: Human mesenchymal stem cells (hMSCs) derived from human mesenchymal stem cells 엑소좀Exosome -모사 나노베지클(- Mosaic nano bezle ( exosomeexosome -mimetic -mimetic nanovesiclesnanovesicles , , NVsNVs )의 분석) Analysis
(1) 투과 전자 현미경(Transmission Electron Microscopy; TEM) (1) Transmission Electron Microscopy (TEM)
글로-방전 탄소-코팅 구리 그리드(glow-discharged carbon-coated copper grids)(Electron Microscopy Sciences, Fort Washington, PA)에 정제된 NVs을 가하였다. NVs가 1시간 동안 그리드 상에 흡수되도록 한 후, 그리드를 4% 파라포름알데하이드로 10분간 고정시켰으며 탈이온수의 물방울로 세척한 다음, 2% 우라닐 아세테이트(Ted Pella, Redding, CA)로 음성 염색(negative stain)하였다. 전자 현미경 사진은 100 kV의 가속 전압에서 JEM 1011 microscope(JEOL, Tokyo, Japan)로 기록되었다.Purified NVs were added to glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Fort Washington, Pa.). After the NVs were allowed to absorb on the grid for 1 hour, the grid was fixed with 4% paraformaldehyde for 10 minutes, washed with deionized water and then negative with 2% uranyl acetate (Ted Pella, Redding, CA) And stained (negative stain). The electron micrographs were recorded on a JEM 1011 microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 100 kV.
(2) 동적 광 산란법(Dynamic light scattering; DLS) (2) Dynamic light scattering (DLS)
NVs의 크기 분포를 Zetasizer Nano ZS(Malvern Instrument Ltd., Malvern, U.K.)로 측정하였다. 상대적 빈도에 기초한 크기 분포는 10 × 30 s에 대한 산란 강도에서 샘플을 통과하는 적외선(파장 = 633 nm)으로 측정하였다.The size distribution of NVs was measured with a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, U.K.). The size distribution based on the relative frequency was measured with an infrared ray (wavelength = 633 nm) passing through the sample at a scattering intensity of 10 x 30 s.
시험예 3. 인간 중간엽 줄기세포(Human mesenchymal stem cells, hMSCs) 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles, NVs) 효능의 인 비보(Test Example 3: Exosome-mimetic nanovesicles (NVs) derived from human mesenchymal stem cells (hMSCs) in in vivovivo ) 분석) analysis
(1) Matrigel plug assay (1) Matrigel plug assay
상기 제조한 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 및 면역세포 침윤 효과를 확인하기 위해서, 세포 외 기질 성분 복합체인 Matrigel(0.5mL)에 엑소좀-모사 나노베지클(10 μg)을 섞어 6주령 수컷 마우스에 피하 주사하고, 7일 후에 주입한 Matrigel을 얻어 면역형광염색법을 수행하였다. 양성 대조군으로는 VEGF(250 ng)을 사용하였다. 내피세포 마커인 CD31과 대식세포 마커인 F4/80를 염색하였고, Hoechst을 활용하여 세포의 핵을 염색한 후, 공초점 현미경을 사용하여 이미지를 관찰하였다(도 4 참조).To confirm the neovascularization and immune cell infiltration effect of the prepared mesenchymal stem cell-derived exosome-simulated nano-beadlets, Matrigel (0.5 mL) of the extracellular matrix component complex was administered with exosome-mimosa nano-bezule 10 μg) was injected subcutaneously into 6-week-old male mice, and Matrigel injected after 7 days was obtained and immunofluorescence staining was performed. VEGF (250 ng) was used as a positive control. The endothelial cell marker CD31 and the macrophage marker F4 / 80 were stained. Hoechst was used to stain the nuclei of the cells, and images were observed using a confocal microscope (see FIG. 4).
시험예 4. 인간 중간엽 줄기세포(Human mesenchymal stem cells, hMSCs) 유래의 엑소좀-모사 나노베지클(exosome-mimetic nanovesicles, NVs) 효능의 인 비트로(Test Example 4. Exosome-mimetic nanovesicles (NVs) derived from human mesenchymal stem cells (hMSCs) in vitroin vitro ) 분석) analysis
(1) Proliferation assay(WST-1) (1) Proliferation assay (WST-1)
인간 내피세포(Human microvascular endothelial cell, HMEC-1)를 96-웰 플레이트에 5 × 104 cells/well씩 분주하여 배양하고, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 다양한 농도(0, 0.1, 1, 10 μg/mL)로 처리한 후 24시간 동안 추가 배양하였다. Washing 과정 없이, 배양액에 WST-1 시약을 10 μL/well로 첨가한 후 4시간 동안 37 ℃에서 배양하고 420-480 nm의 파장에서 흡광도를 측정하였다. 양성 대조군으로 VEGF(10 ng/mL)를 사용하였다.Human endothelial cells (HMEC-1) were cultured in a 96-well plate at a density of 5 × 10 4 cells / well, and the mesenchymal stem cells derived exosome-simulated nano-beads were cultured at various concentrations (0 , 0.1, 1, 10 μg / mL) and then further cultured for 24 hours. Without washing, WST-1 reagent was added to the culture solution at 10 μL / well, incubated at 37 ° C. for 4 hours, and absorbance was measured at a wavelength of 420-480 nm. VEGF (10 ng / mL) was used as a positive control.
(2) Migration assay(Boyden chamber assay) (2) Migration assay (Boyden chamber assay)
폴리카보네이트 멤브레인(8-μm pore: 대식세포, 12-μm pore: 내피세포)을 0.1% 젤라틴으로 코팅하였다. 세포는 5 μM의 5-chloromethylfluorescein diacetate(CMFDA)가 포함된 serum-free media로 30분 동안 37 ℃에서 배양하여 세포질을 염색한 후, 염색이 끝난 세포는 새로운 serum-free media로 재현탁시켰다. 아래쪽 챔버에 CMFDA로 염색된 세포를 3 × 104 cells/well로 분주하고, 그 위에 젤라틴으로 미리 코팅한 멤브레인을 올렸다. 이후, 챔버를 뒤집어서 2시간 동안 37 ℃의 인큐베이터에서 배양한 다음, 다시 챔버를 뒤집어서 위쪽 챔버에 처리하고자 하는 다양한 농도의 엑소좀-모사 나노베지클 또는 세포 배양액(CM)을 넣은 후, 뒤집어서 2시간 동안 37 ℃의 인큐베이터에서 추가 배양하였다. 세포의 이동이 끝나면, 70% ice-cold 에탄올을 이용하여 고정시킨 후, 형광현미경으로 관찰하였다. 양성 대조군으로는 VEGF(10 ng/mL) 및 EGM2-MV를 사용하였다.Polycarbonate membranes (8-μm pore: macrophages, 12-μm pore: endothelial cells) were coated with 0.1% gelatin. Cells were stained cytoplasmically with 5 μM 5-chloromethylfluorescein diacetate (CMFDA) in serum-free media for 30 min at 37 ° C, and the stained cells were resuspended in fresh serum-free media. Cells stained with CMFDA in the lower chamber were dispensed at 3 × 10 4 cells / well, and a membrane pre-coated with gelatin was placed thereon. Thereafter, the chamber was turned upside down and incubated for 2 hours in an incubator at 37 ° C. Then, the chamber was turned upside down and various concentrations of exosome-simulated nano-beads or cell culture medium (CM) Lt; RTI ID = 0.0 > 37 C < / RTI > After cell migration, cells were fixed with 70% ice-cold ethanol and observed with fluorescence microscope. VEGF (10 ng / mL) and EGM2-MV were used as positive control groups.
(3) Preparation of conditioned medium(CM) (3) Preparation of conditioned medium (CM)
마우스 유래 대식세포(RAW264.7)를 배양하고, 다양한 농도(0, 0.01, 0.1, 1 μg/mL)의 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 처리하여 24시간 동안 배양하였다. 죽은 세포 등을 제거하기 위해, 500 ×g, 5분간 원심분리를 한 후 0.22 μm filtration을 통해 대식세포 유래 세포 배양액(CM)을 얻었다.The mouse-derived macrophages (RAW 264.7) were cultured and treated with various concentrations (0, 0.01, 0.1, 1 μg / mL) of mesenchymal stem cell-derived exosome-simulated nano-beads for 24 hours. To remove dead cells and the like, the cells were centrifuged at 500 × g for 5 minutes, and cultured with macrophages (CM) through 0.22 μm filtration.
(4) RNA isolation and Real-time PCR (4) RNA isolation and Real-time PCR
Trizol을 이용하여 세포의 RNA를 분리한 후, High capacity RNA to cDNA kit(AB Applied Biosystems)를 활용하여 cDNA을 만들었다. 각 cDNA(30ng)는 One Step SYBR RT-PCR Kit(TaKaRa Bio)를 활용하여 LightCycler 2.0 PCR system(Roche Diagnostics)으로 real-time PCR을 진행하였다. 이때, 사용한 프라이머의 종류는 다음과 같다.Trizol was used to isolate RNA from cells and cDNA was prepared using high capacity RNA to cDNA kit (AB Applied Biosystems). Each cDNA (30 ng) was subjected to real-time PCR using the LightCycler 2.0 PCR system (Roche Diagnostics) using the One Step SYBR RT-PCR Kit (TaKaRa Bio). Here, the types of primers used are as follows.
서열번호 1 : hKGF Forward 5'-AAAGGGGATTCCTGTAAGAG-3'SEQ ID NO: 1: hKGF Forward 5'-AAAGGGGATTCCTGTAAGAG-3 '
서열번호 2 : hKGF Reverse 5'-TGCTGGAACTGGTTCTTTAT-3'SEQ ID NO: 2: hKGF Reverse 5'-TGCTGGAACTGGTTCTTTAT-3 '
서열번호 3 : hTGF-α Forward 5'-TGTTATCTTCAAGCCAGGTT-3'SEQ ID NO: 3: hTGF-α Forward 5'-TGTTATCTTCAAGCCAGGTT-3 '
서열번호 4 : hTGF-α Reverse 5'-GGTCTCCTGAGCAGTGTTAG-3'SEQ ID NO: 4: hTGF-α Reverse 5'-GGTCTCCTGAGCAGTGTTAG-3 '
서열번호 5 : hGAPDH Forward 5'-CGAGATCCCTCCAAAATCAA-3' SEQ ID NO: 5: hGAPDH Forward 5'-CGAGATCCCTCCAAAATCAA-3 '
서열번호 6 : hGAPDH Reverse 5'-TTCACACCCATGACGAACAT-3'SEQ ID NO: 6: hGAPDH Reverse 5'-TTCACACCCATGACGAACAT-3 '
시험결과 1. Test results 1. 중간엽Intermediate lobe 줄기세포 유래의 Stem cell-derived 엑소좀Exosome -모사 -copy 나노베지클의Nano-vesicle 제조 및 분석 결과 Manufacturing and analysis results
중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 제조하기 위해서, 세포를 passage 6까지 계대배양하고 신생 혈관 생성 효과를 증가시키기 위해서, TNF-α(20 ng/mL)를 처리하여 12시간 추가 배양하여 엑소좀-모사 나노베지클을 제조하였으며, 도 2a 및 2b에서 보는 바와 같이 제조된 엑소좀-모사 나노베지클의 형태와 크기를 투과 전자 현미경 및 동적 광산란 분석을 통해 확인하였다. 제조된 엑소좀-모사 나노베지클은 47.55 ± 5.03 nm의 평균 직경을 갖는 것으로 나타났다.In order to prepare exosome-mimic nano-beads from mesenchymal stem cells, TNF-α (20 ng / mL) was added to the cells to pass the passage 6 until
또한, 동일한 배양 조건에서 분리 및 정제한 기존의 중간엽 줄기세포 유래의 엑소좀과 본 명세서에 따른 엑소좀-모사 나노베지클의 수득률을 비교하면, 1개의 150 mm 배양 접시를 기준으로 엑소좀-모사 나노베지클은 엑소좀에 비해 단백질 양으로는 약 40배, 입자수로는 약 150 배 많은 양을 얻을 수 있음을 확인하였다(도 3 참조). 따라서, 종래 분리 및 정제가 어렵고 수득률이 매우 낮아 그 활용이 어려운 중간엽 줄기세포 유래의 엑소좀이 갖는 문제를 해결할 수 있다.Further, when comparing the yields of existing mesenchymal stem cell-derived exosomes isolated and purified under the same culture conditions and the exosome-sima nano bezle according to the present invention, it was found that exosome- It was confirmed that the simulated nano beads can obtain about 40 times as much protein and about 150 times as much as the particle size of exosomes (see FIG. 3). Therefore, it is possible to solve the problem of exosome originating from mesenchymal stem cells, which has been difficult to separate and purify in the past and has a very low yield.
시험결과 2. 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 효과Test results 2. Neovascularization effect of exosome-mimosa nano-vesicles derived from mesenchymal stem cells
(1) 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 및 면역세포 침윤 효과 (1) Neovascularization and immune cell infiltration effect of exosome-mimosa nano-beads from mesenchymal stem cells
중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 및 면역세포 침윤 효과를 확인하기 위해서, 세포 외 기질 성분 복합체인 Matrigel에 엑소좀-모사 나노베지클을 섞어 마우스에 피하 주사하고 7일 뒤 주입한 Matrigel을 얻어 면역형광염색법을 시행하였다.To confirm the neovascularization and immune cell infiltration effects of exosome-mimosa nano-beadlets derived from mesenchymal stem cells, Matrigel, which is an extracellular matrix component complex, was subcutaneously injected into mice and mixed with exosome- Matrigel was injected and immunofluorescent staining was performed.
내피세포 마커로 잘 알려진 CD31을 활용하여 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 효과를 확인한 결과, 도 5에서 보는 바와 같이 대조군(PBS)에 비해 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클(10 μg) 그룹은 Matrigel 내부에 새로운 혈관을 많이 생성하였으며, 이는 기존에 혈관 관련 연구에서 많이 사용하는 양성 대조군인 VEGF(250 ng)와 유사한 효과임을 확인하였다. 특히, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클 그룹은 VEGF 그룹보다 곧고 길쭉하며, 끊어짐이 적은 혈관을 더 많이 관찰할 수 있었다.As shown in FIG. 5, the effect of exo-soma nano-beadlets derived from mesenchymal stem cells on the expression of mesenchymal stem cell-derived cells compared to the control (PBS) The exosome-sima nano-vesicle (10 μg) group produced a lot of new blood vessels inside the Matrigel, confirming that it is similar to the positive control group VEGF (250 ng), which has been widely used in vascular studies. In particular, the exosome - mimosa nano - bequick group derived from mesenchymal stem cells was more straight and elongated than the VEGF group, and more blood vessels with less breakage were observed.
또한, 신생 혈관이 생성된 주변의 면역세포 침윤 정도를 확인하기 위해서, 대식세포 마커인 F4/80을 활용하여 Matrigel 절편을 염색한 결과, 도 6에서 보는 바와 같이 CD31 염색 결과와 유사하게 PBS 그룹에 비해 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클(10 μg) 그룹에서 Matrigel 내로 많은 수의 대식세포가 침윤되었음을 확인하였다. 이는 양성 대조군인 VEGF(250 ng)와 유사한 효과였다. 더불어, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클 그룹과 달리 VEGF 그룹은 침윤된 대식세포가 새롭게 생성된 혈관 주변(흰색 실선)에 더 많이 모여 있는 현상이 관찰되었다. 이러한 현상은 혈관 및 혈관 주변에 손상 또는 그로 인한 재생 등이 일어나는 경우에 관찰되는 것으로서(Int J Dev Biol., 55(4-5):495-503, 2011), VEGF에 의해 새롭게 생성된 혈관이 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클에 의해 생성된 혈관에 비해 건강하지 않음을 간접적으로 알 수 있었다.In order to confirm the degree of infiltration of the immune cells around the new blood vessels, Matrigel slices were stained using F4 / 80 macrophage marker. As a result, as shown in FIG. 6, (10 μg) group of mesenchymal stem cells derived mesenchymal stem cells showed a large number of macrophages infiltrated into Matrigel. This was similar to that of the positive control, VEGF (250 ng). In addition, unlike the exosome-mimosa nano-vesicle group derived from mesenchymal stem cells, the VEGF group was observed to accumulate more around the newly formed blood vessels (white solid line) of infiltrated macrophages. This phenomenon is observed in cases where damage or restoration occurs around blood vessels and blood vessels (Int J Dev Biol., 55 (4-5): 495-503, 2011), and newly generated blood vessels by VEGF It was indirectly found that the cells were not as healthy as the blood vessels produced by mesenchymal nano-beads from mesenchymal stem cells.
결론적으로, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 신생 혈관 생성 및 면역세포 침윤 효과를 확인하였다.In conclusion, neovascularization and immune cell infiltration effects of exosome-mimosa nano-beads from mesenchymal stem cells were confirmed.
(2) 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 내피세포 증식 및 이동에 미치는 영향 (2) Effect of mesenchymal stem cells on the proliferation and migration of endothelial cells
중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 Matrigel 내부에 새로운 혈관을 생성하는 효과가 내피세포의 증식 또는 이동에 의한 효과인지 확인하기 위해서, 인간 내피세포(Human microvascular endothelial cell, HMEC-1)를 배양하고 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 다양한 농도(0, 0.1, 1, 10 μg/mL)로 처리한 후 내피세포의 증식 및 이동 효과를 확인하였다.In order to confirm whether the effect of producing new blood vessels inside the matrix of exosome-mimosa nano-beadlets derived from mesenchymal stem cells was due to the proliferation or migration of endothelial cells, human endothelial cells (HMEC-1 ) Were cultured and treated with various concentrations (0, 0.1, 1, 10 μg / mL) of exosome-mimosa nano-beads from mesenchymal stem cells, and proliferation and migration effect of endothelial cells were confirmed.
WST-1 proliferation assay로 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 처리된 내피세포의 증식률을 확인한 결과, 다양한 농도의 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클 처리 여부와 상관 없이 내피세포의 증식 효과는 확인하지 못하였다(도 7의 A 참조). 그러나, boyden chamber assay를 통해 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 처리된 내피세포의 이동 효과를 확인한 결과, 1 μg/mL의 농도에서 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클의 내피세포 이동 효과가 가장 높음을 확인하였다(도 7의 B, C 참조). 양성 대조군으로는 VEGF(10 ng/mL)를 사용하였다.In the WST-1 proliferation assay, the proliferation rate of endothelial cells treated with mesenchymal nano beadlets derived from mesenchymal stem cells was examined. As a result, it was found that the exosome- The proliferation effect of endothelial cells was not confirmed (see Fig. 7A). However, by examining the migration effect of endothelial cells treated with mesenchymal nano beadlets derived from mesenchymal stem cells through the boyden chamber assay, it was found that at a concentration of 1 μg / mL, the exosome- It was confirmed that the endothelial cell migration effect of vesicle was the highest (see B and C in Fig. 7). VEGF (10 ng / mL) was used as a positive control.
(3) 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 대식세포 이동에 미치는 영향 (3) Effects of exosome-mimosa nano-beadlets derived from mesenchymal stem cells on macrophage migration
중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 신생 혈관 생성 지역으로 면역세포 침윤을 직접적으로 유도하는지 확인하기 위해서, 마우스 대식세포(Raw264.7)를 배양하고 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 다양한 농도(0, 0.01, 0.1, 1 μg/mL)로 처리한 후 대식세포의 이동 효과를 확인하였다.In order to confirm whether the exosome-simulated nano-beadlets derived from mesenchymal stem cells directly induce immune cell infiltration into the neovascularization area, mouse macrophages (Raw 264.7) were cultured and the mesenchymal stem cell-derived exosomes - The effect of macrophages was examined after treatment with various concentrations (0, 0.01, 0.1, 1 μg / mL) of the simulated nano-beads.
Boyden chamber assay를 통해 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 처리된 대식세포의 이동 효과를 확인한 결과, 기존과 유사하게 1 μg/mL의 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 처리된 대식세포가 가장 많이 이동하였으며, 양성 대조군으로 처리한 VEGF(10 ng/mL)보다 더 많은 대식세포 이동 유도 효과가 있음을 확인할 수 있었다(도 8 참조).In the Boyden chamber assay, the migration effect of macrophage-treated macrophage-mediated macrophage-mediated macrophage-mediated migration of macrophage stem cells was examined. As a result, 1 μg / mL mesenchymal stem cell-derived exosome- The vesicle-treated macrophages migrated the most, indicating that macrophage migration was more effective than VEGF (10 ng / mL) treated with the positive control (see FIG. 8).
결과를 종합해 보면, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클은 내피세포와 대식세포의 이동을 유도하여 신생 혈관을 형성하는 효과가 있음을 알 수 있었다. 이에 따라, 세포의 손상 등의 증상 완화 및 개선 효과를 기대할 수 있음을 확인하였다.The results suggest that exosome-mimosa nano-beads derived from mesenchymal stem cells induce the migration of endothelial cells and macrophages to form neovascularization. Thus, it was confirmed that symptoms such as cell damage and the like can be expected to be alleviated and improved.
(4) 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클로 처리된 대식세포가 내피세포 이동에 미치는 영향 (4) Effect of mesenchymal stem cells on endothelial cell migration by exosome-mimic nanobegyclo-treated macrophages
중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 대식세포를 통해 간접적으로 내피세포의 이동에 미치는 영향을 확인하기 위해, 마우스 대식세포(Raw264.7)를 배양하고 다양한 농도(0, 0.01, 0.1, 1 μg/mL)의 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 처리하여 24시간 동안 배양한 후 세포 배양액(conditioned medium, CM)을 얻었다. 세포 배양액은 상기와 동일한 시험 방법을 통해 내피세포의 이동 효과를 확인하였다.To confirm the effect of mesenchymal nano beadlets derived from mesenchymal stem cells on the migration of endothelial cells indirectly through macrophages, mouse macrophages (Raw 264.7) were cultured and cultured at various concentrations (0, 0.01, 0.1, and 1 μg / mL) of the mesenchymal stem cell-derived exosome-mimosa nano-beadlets were cultured for 24 hours and then conditioned medium (CM) was obtained. The cell culture medium was confirmed to have endothelial cell migration effect through the same test method as described above.
Boyden chamber assay를 통해 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클로 처리된 대식세포 세포 배양액의 내피세포 이동 효과를 확인한 결과, 기존과 유사하게 1 μg/mL의 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 처리된 대식세포에서 얻은 세포 배양액에서 내피세포 이동 효과가 가장 높은 것을 확인하였다(도 9 참조). 또한, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클을 내피세포에 직접 처리한 것과 유사 혹은 더 우수한 효과가 있음을 알 수 있었다. 양성 대조군으로는 VEGF(10 ng/mL)를 사용하였다.The endothelial cell migration of mesenchymal stem cells from mesenchymal stem cells was examined by Boyden chamber assay. As a result, 1 μg / ml mesenchymal stem cell-derived exosomes - The endothelial cell migration effect was highest in the cell culture obtained from macrophage-treated macrophages (see FIG. 9). In addition, it was found that the exosome-mimosa nano-beadlet derived from mesenchymal stem cells had a similar or better effect than that directly treated with endothelial cells. VEGF (10 ng / mL) was used as a positive control.
이에 따라, 중간엽 줄기세포 유래의 엑소좀-모사 나노베지클이 생체 내에서 면역세포(대식세포 등)의 침윤을 유도하고, 이들에 의해 내피세포의 이동이 유도 또는 촉진됨으로써 최종적으로 신생 혈관 형성에 영향을 미치는 것을 알 수 있었다.As a result, mesenchymal nano beques derived from mesenchymal stem cells induce infiltration of immune cells (macrophages, etc.) in vivo, and induction or promotion of migration of endothelial cells by them induces ultimately neovascularization . The results of this study are as follows.
본 발명의 일 측면에 따른 조성물의 제형예를 아래에서 설명하나, 다른 여러 가지 제형으로도 응용 가능하며, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Formulation examples of compositions according to one aspect of the present invention are described below, but may be applied to various other formulations, which are not intended to be limiting but merely illustrative of the invention.
[제형예 1] 주사제[Formulation Example 1]
엑소좀-모사 나노베지클 50 mg, 주사용 멸균 증류수 적량, pH 조절제 적량을 혼합하고, 통상의 주사제의 제조방법에 따라 1 앰플당(2㎖) 상기의 성분 함량으로 제조하였다.50 mg of exosome-sima nano bezicel, sterilized distilled water suitable for injection, and pH adjuster were mixed, and the contents were adjusted to the above contents in the amount of 2 ml per ampoule according to the usual injection preparation method.
[제형예 2] 연질캅셀제[Formulation Example 2] Soft capsule
엑소좀-모사 나노베지클 50mg, L-카르니틴 80~140mg, 대두유 180mg, 팜유 2mg, 식물성 경화유 8mg, 황납 4mg 및 레시틴 6mg을 혼합하고, 통상의 방법에 따라 1캡슐 당 400mg씩 충진하여 연질캅셀제를 제조하였다.400 mg of the exosome-
[제형예 3] 정제[Formulation Example 3] Tablets
엑소좀-모사 나노베지클 50mg, 갈락토올리고당 200mg, 유당 60mg 및 맥아당 140mg을 혼합하고 유동층 건조기를 이용하여 과립한 후 당 에스테르(sugar ester) 6mg을 첨가하여 타정기로 타정하여 정제를 제조하였다.50 mg of exosome-sima nano bezicel, 200 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed and granulated using a fluidized bed drier, followed by addition of 6 mg of sugar ester and tabletting by tableting.
[제형예 4] 과립제[Formulation Example 4]
엑소좀-모사 나노베지클 50mg, 무수결정 포도당 250mg 및 전분 550mg을 혼합하고, 유동층 과립기를 사용하여 과립으로 성형한 후 포에 충진하여 과립제를 제조하였다.50 mg of exosome-sima nano bezicel, 250 mg of anhydrous crystalline glucose and 550 mg of starch were mixed and granulated into granules using a fluidized bed granulator and filled in a bag to prepare granules.
[제형예 5] 드링크제[Formulation Example 5]
엑소좀-모사 나노베지클 50mg, 포도당 10g, 구연산 0.6g, 및 액상 올리고당 25g을 혼합한 후 정제수 300ml를 가하여 각 병에 200ml씩 충진하였다. 병에 충진한 후 130℃에서 4~5초간 살균하여 드링크제 음료를 제조하였다.50 mg of exosome-sima nano bezicel, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide were mixed and 300 ml of purified water was added to each bottle to fill 200 ml of each bottle. And then sterilized at 130 DEG C for 4 to 5 seconds to prepare a beverage drink.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> AMOREPACIFIC CORPORATION
POSTECH ACADEMY-INDUSTRY FOUNDATION
<120> COMPOSITION FOR PROMOTING ANGIOGENIC ACTIVITY COMPRISING
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<120> COMPOSITION FOR PROMOTING ANGIOGENIC ACTIVITY COMPRISING
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Claims (14)
A composition for promoting angiogenesis, comprising exosome-mimetic nanovesicles derived from adult stem cells as an active ingredient.
상기 엑소좀-모사 나노베지클은 성체줄기세포를 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시킨 성체줄기세포의 여과물에 포함된 나노 크기의 베지클인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
The exosome-mimetic nano-beadlet is a nano-sized vesicle that is contained in a filtrate of adult stem cells sequentially passed through a large-size filter and a small-size filter in two or more different size membrane filters of adult stem cells , A composition for promoting angiogenesis.
상기 엑소좀-모사 나노베지클은 30 내지 200 nm의 직경을 갖는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein the exosome-simulated nano-beads have a diameter of 30 to 200 nm.
상기 엑소좀-모사 나노베지클은 성체줄기세포의 여과물로부터 10% 및 50% 농도의 이오딕사놀을 이용한 밀도 구배 초원심분리 시, 10% 농도의 이오딕사놀과 50% 농도의 이오딕사놀 사이에 위치하는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
The exosome-simulated nano-beadlets were obtained from the filtrate of adult stem cells by density gradient ultracentrifugation using 10% and 50% concentration of iodixanol at a concentration of 10% of iodixanol and a concentration of 50% of iodixanol Wherein the composition for promoting angiogenesis is placed between the first and second vessels.
상기 성체줄기세포는 골수, 제대혈, 혈액, 피부, 지방 및 태반으로 이루어진 군에서 선택되는 1 이상으로부터 유래된 중간엽 줄기세포인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein said adult stem cells are mesenchymal stem cells derived from at least one selected from the group consisting of bone marrow, umbilical cord blood, blood, skin, fat and placenta.
상기 성체줄기세포는 TNF-α를 처리하여 배양한 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein said adult stem cells are cultured by treatment with TNF- ?.
상기 성체줄기세포는 passage 6까지 계대배양한 후 TNF-α를 처리하여 배양한 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein said adult stem cells are subcultured to passage 6 and then cultured by treatment with TNF- ?.
상기 멤브레인 필터는 1 내지 10 ㎛ 크기의 평균 공극을 갖는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein the membrane filter has an average pore size of 1 to 10 mu m.
상기 초원심분리는 100,000 ×g 이상에서 수행하는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein the ultracentrifugation is performed at 100,000 x g or more.
상기 조성물은 혈관 내피세포의 이동을 유도 또는 증가시키는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein said composition induces or increases the migration of vascular endothelial cells.
상기 조성물은 혈관 신생이 요구되는 질환에서 혈관 신생을 촉진하는 것인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein the composition promotes angiogenesis in a disease requiring angiogenesis.
상기 혈관 신생이 요구되는 질환은 상처, 화상, 궤양, 허혈, 동맥경화증, 협심증, 심근경색, 뇌혈관성 질환 및 탈모증으로 이루어진 군에서 선택되는 1 이상인, 혈관 신생 촉진용 조성물.
12. The method of claim 11,
Wherein the disease requiring angiogenesis is at least one selected from the group consisting of wound, burn, ulcer, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease and alopecia.
상기 조성물은 약학 조성물 또는 식품 조성물인, 혈관 신생 촉진용 조성물.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition or a food composition.
(1) 성체줄기세포를 배양하는 단계;
(2) 배양된 성체줄기세포를 수확하여 완충용액에 현탁시키는 단계;
(3) 상기 현탁액을 2개 이상의 크기가 다른 멤브레인 필터로 크기가 큰 필터에서 작은 필터 순으로 순차적으로 통과시켜 여과물을 제조하는 단계; 및
(4) 상기 여과물로부터 초원심분리를 통해 엑소좀-모사 나노베지클을 수득하는 단계;를 포함하는 성체줄기세포 유래의 엑소좀-모사 나노베지클을 제조하는 방법.14. A method for producing exosome-mimetic nanobicles from adult stem cells according to any one of claims 1 to 13,
(1) culturing adult stem cells;
(2) harvesting the cultured adult stem cells and suspending them in a buffer solution;
(3) passing the suspension through two or more size different membrane filters in order from a large filter to a small filter in order to produce a filtrate; And
(4) obtaining an exosome-mimetic nanobeads from the filtrate by ultracentrifugation; and (4) culturing the exosome-mimetic nanobeads from the adult stem cells.
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| PCT/KR2017/005965 WO2018004143A1 (en) | 2016-06-30 | 2017-06-08 | Angiogenesis-promoting composition containing adult stem cell-derived exosome-mimetic nanovesicle |
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| WO2019151744A1 (en) * | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | Adult stem cell-derived nanovesicles and use thereof for targeted therapy |
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| KR20210029688A (en) * | 2019-09-06 | 2021-03-16 | 주식회사 엠디뮨 | Composition for preventing or treating salivary gland disease comprising cell derived vesicles |
| WO2021141207A1 (en) * | 2020-01-09 | 2021-07-15 | 주식회사 엠디뮨 | Cell extruder and cell extrusion method |
| WO2022169258A1 (en) * | 2021-02-02 | 2022-08-11 | 주식회사 엠디뮨 | Cell-derived vesicles with increased cellular uptake capacity and method for producing same |
| WO2022231347A1 (en) * | 2021-04-28 | 2022-11-03 | 인천대학교 산학협력단 | Freeze-dry protective agent for extracellular vesicles |
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